1. Field of the Invention
This invention relates to the construction of a genetically engineered vector which would be useful as a vaccine against poultry diseases or for ferrying desirable genes into the avian system. There are over 4 billion chickens and 200 million turkeys raised annually in the United States alone. On the average, each bird is vaccinated against 10 different diseases. There are several problems associated with conventional vaccines in current use against poultry diseases. Killed or subunit vaccines, which are necessary for some pathogens, are safe but relatively inefficient. The live vaccines are typically more effective, but occasionally exhibit undesirable pathogenic effects. Another problem with current poultry vaccines is that they are susceptible to vaccine breaks; that is, the current vaccines do not provide protection against new, highly virulent strains of pathogens. Finally, some pathogens such as avian influenza have no existing vaccine, and the pathogenic nature of the infectious agent has precluded the development of either a live or killed virus vaccine.
In light of the problems with conventional vaccines, there are some obvious advantages of developing a genetically engineered vector which would be useful as a vaccine. Such a vector could be constructed which lacked or contained only a portion of any viral genes responsible for pathogenesis. This would enable protection without the risk of disease induction. In addition, polyvalent, live recombinant DNA vaccines expressing several different foreign genes could be easily constructed. These should be less expensive to prepare and administer than multiple current subunit or attenuated vaccines. Most importantly, rDNA vaccines could be constructed that are more efficacious than existing vaccines. By using the proper promoters, a live rDNA vaccine could be induced to express a higher level of immunogenic proteins than could be obtained with conventional attenuated vaccines, thereby more strongly stimulating a protective response in the host immune system. Live rDNA vaccines will stimulate both humoral and cell mediated immunity, unlike subunit vaccines that only stimulate humoral immunity.
Some of the avian herpesviruses, particularly Marek's disease virus (MDV), are the cause of economically important diseases in poultry. As a result of the economic attention given to MDV, it has been the subject of extensive scientific investigation in recent years. Consequently, there exist successful live virus vaccines for MDV which are logical candidates as source materials for developing a suitable vector. The MDV's are typically characterized by a double stranded DNA genome of approximately 150 to 180 kb and are classified as gamma-herpesviruses.
There are three distinct serotypes of MDV found in chickens: (1) serotype 1, an oncogenic virus which induces a T cell lymphoma in chickens and includes high- and low-virulent MDV and their attenuated variants; (2) serotype 2, a nononcogenic MDV; and (3) serotype 3, herpesvirus of turkeys (HVT). Neoplasms (lymphomas) induced by serotype 1 MDV can be prevented by vaccination with live virus preparations of serotype 2 and/or HVT. Although cross-reactive proteins have been detected, the three serotypes have unique DNA restriction patterns. The extent of DNA homology between the serotypes has been reported from as low as 5% to approaching 70%.
One difficulty in using MDV as a possible expression vector has been the failure so far to identify sequences which do not appear to be essential for replication of MDV as sites for the insertion of foreign genes. The thymidine kinase (tk) gene which has been used in other herpesviruses as an insertion site has not been identified in MDV. Screening for other sites is confounded by the absence of a positive selection system and the potential for lack of stability in regard to insertion and expression of any DNA. Also, there is a lack of basic knowledge of MDV, particularly with respect to promoters and transcriptional regulation.
2. Description of the Prior Art
Defective virus particles are often seen as a result of serial undiluted passage (high multiplicity of infection) of virus stocks of many types of viruses. Many herpesviruses have been observed to contain defective particles after serial propagation in vitro. N. Frenkel [Defective Interfering Herpesviruses, In The Human Herpesviruses--An Interdisciplinary Prospective (eds. A. J. Nahmias, W. R. Dowdle, and R. S. Schinazy), pp. 91-120, Elsevier-North Holland, Inc., New York, (1981)] has reported on two classes of defective viruses which contain different origins of replication in herpes simplex virus (HSV) stocks. These defective viruses have been shown to be head-to-tail reiterations (repeat units) of specific HSV DNA sequences (seed) which are amplified to form concatemers of approximately the full length of wild type virus [150 kilobases (kb)] and repackaged as defective particles [Spaete et al., Cell 30: 295-304 (1982)]. These defectives, termed amplicons, have been shown to carry cis-acting signals, an origin of replication and a packaging signal. The origin of replication and packaging signal allow replication and encapsidation of the defective particle when trans-acting functions are provided by a competent helper virus [Spaete et al., Proc. Natl. Acad. Sci. USA 82: 694-698 (1985); Vlazny et al., Proc. Natl. Acad. Sci. USA 78: 742-746 (1981)].
Yates et al. [Nature 313: 812-815 (1985)] reported a stable plasmid consisting of the ori P and EBNA-1 of Epstein-Barr virus with a selectable gene which persists at 1-3 copies/cell. This replicon is persistent and stable with serial passage (Yates et al. supra).
Amplicons have been shown to replicate and express foreign DNA sequences which have been engineered into the seed. Frenkel et al. [Eukaryotic Viral Vectors (ed. Y. Gluzman), Cold Spring Harbor (1982)] and Spaete et al. [Cell 30: 295-304 (1982)] reported on the construction of a chimeric amplicon by cloning a 3- to 8-kb repeat unit from HSV-1 defective genomes into a derivative of a bacterial plasmid. Cotransfection of cells with the amplicon and a helper virus resulted in the regeneration of chimeric defective genomes containing multiple reiterations of the seed DNA sequences containing repeat units in which the bacterial plasmid sequences were linked to the amplicon sequences. These chimeric defective genomes were efficiently packaged into structural virions and were structurally stable through multiple serial passaging in eucaryotic cell culture. The results established that foreign DNA sequences can be introduced into defective HSV genome repeat units and be stably propagated in virus populations when a helper virus is present.
Kwong et al. [J. Virol. 51(3): 595-603 (Sept. 1984)] has shown propagation of relatively large sets of eucaryotic DNA sequences within chimeric packaged defective genomes. The largest chimeric genomes tested having an overall size of 19.8 kb comprised a 12-kb DNA sequence encoding the chicken ovalbumin gene, an HSV repeat unit, and bacterial plasmid sequences. These genomes replicated in serial passage but were not as stable as other seed amplicons tested ranging in size from 11 to 15 kb and containing subsets of the 12-kb chicken DNA sequences.
It was later established by Kwong et al. [Virology 142: 421-425 (1985)] that a eucaryotic gene inserted into defective virus genomes could be expressed in HSV-infected cells. Sequences of the chicken ovalbumin gene were fused to an HSV alpha promoter and to genomic ovalbumin 3'-flanking sequences. The chimeric alpha-ovalbumin gene was introduced into defective HSV genomes which were stably propagated in serially passaged virus stocks in the presence of helper virus. The chimeric gene was abundantly expressed in cells infected with the resultant virus stocks.